PURIFICATION AND CHARACTERIZATION OF MONOMERIC ESCHERICHIA-COLI VITAMIN-B-12 RECEPTOR WITH HIGH-AFFINITY FOR COLICIN-E3

The btuB gene product from Escherichia coli is a 66.5-kDa integral out er membrane protein required for high-affinity uptake of cyanocobalami n and the translocation of E group colicins and colicin A. Efficient p urification of overexpressed BtuB containing stoichiometric levels of bound lipopolysaccharide has been achieved through the extraction of t he outer membrane with nonionic detergent followed by ion-exchange chr omatography. Analysis of far UV circular dichroism spectra indicates a predominantly beta-sheet secondary structure (76 +/- 4%) with a low a lpha-helical content (15 +/- 3%), providing the first direct evidence for secondary structure models derived from sequence and hydropathy an alysis. Characterization of the octylglucoside-solubilized receptor by sedimentation equilibrium and sedimentation velocity analysis reveals a monodisperse protein-detergent complex of approximately 89 kDa with a sedimentation coefficient of 4.7 S which, after correction for boun d detergent, indicates that BtuB is purified as a monomer. BtuB binds vitamin B-12 with a stoichiometry of approximately 1:1, as observed by a shift in the sedimentation profile of the vitamin to the much faste r velocity observed for the protein-detergent complex. The preincubati on of colicin E3 with stoichiometric levels of BtuB protects susceptib le strains from the lethal effects of the colicin and results in a com plex with a sedimentation coefficient appropriate for a BtuB-detergent -colicin E3 complex, demonstrating that monomeric BtuB retains high af finity for this particular ligand after isolation.